Myogenesis is a tightly regulated process that involves the sequential activation of regulatory genes, exemplified by the MyoD family of basic helix-loop-helix genes and their downstream targets. We have previously studied the role of Notch signaling in postnatal myogenesis. We began to examine the role of the Deltex family of proteins, a family whose homologue in Drosophila is a positive regulator of Notch signaling. In preliminary studies, we found that Deltex2 inhibits MyoD expression and myogenic differentiation, and that this may occur in a Notch-independent manner. Intriguingly, in Deltex2 expressing cells, there is an enrichment of Histone 3 demethylated at lysine 9 (H3K9me2) at an important regulatory domain of the MyoD promoter. H3K9me2 is known to be associated with repression of gene expression. Furthermore, we found in an unbiased, yeast two hybrid screen that Deltex2 binds to an H3K9 demethylase (JmjD1C). Together, these preliminary data suggest that Deltex2 may play an important role in the control of myogenesis, and that the mechanism may involve a novel function in histone modification and chromatin structure. The focus of this proposal is to explore the role of Deltex in myogenesis and to examine the transcriptional program mediated by the Notch signaling pathway in the regulation of differentiation. There are three Specific Aims.
In Aim 1, we will characterize the pattern of expression and cellular localization of Deltex proteins in myogenic differentiation of primary myoblasts and C2C12 myoblasts. We will further test the functional role of these proteins using both overexpression and knockdown approaches.
In Aim 2, we will use microarray analysis to examine the transcriptional program initiated by Notch signaling to inhibit myogenic differentiation. These studies will also explore the Notch-dependent and Notch-independent pathways by which Deltex2 inhibits myogenic differentiation by using myoblasts in which the Notch signaling pathway has been genetically or pharmacologically inhibited. The studies of Aim 3 will focus on our finding that Deltex expression leads to an enrichment of H3K9me2 associated with the MyoD promoter and that the H3K9 demethylase, JmjD1C, is a Deltex2 binding protein. These studies will examine the functional significance and molecular mechanisms of these interactions to test the hypothesis that Deltex2 inhibits myogenesis by inhibiting JmjD1C, thereby promoting the repression of MyoD expression by the association of the H3K9me2 with a key regulatory domain of the MyoD promoter. These studies will explore very fundamental molecular mechanisms of the regulation of myogenic differentiation and will reveal pathways for the control of cell growth and differentiation that will be important for the handling of myogenic progenitors for stem cell therapy for degenerative muscle diseases.

Public Health Relevance

The control of cellular growth and differentiation is a fundamental process responsible for the development, maintenance and regeneration of tissues throughout the body. The studies of this proposal will examine the molecular mechanisms that regulate the transition between growth and differentiation of cells that give rise to mature muscle and that have therapeutic potential for the treatment of degenerative muscle diseases such as muscular dystrophies. The focus of the research will be biochemical pathways (in particular, the "Notch" signaling pathway) that we and others have studied during tissue development and regeneration, how that biochemical pathway is regulated by a relatively newly discovered family of proteins (the "Deltex" family), and how one form of Deltex may regulate the differentiation of muscle cells by a novel mechanism that involves the modification of proteins that bind to DNA.